Abstract

Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NP(o)) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.

Role of the PDZ ligands in the interaction between the STAS and R domains. (a) Extracts were prepared from HEK293 cells transfected with the indicated amount of cDNA encoding CFTR, ΔC-CFTR and mDRA, and used to test for co-immunoprecipitation of mDRA and CFTR. In the experiments with low protein expression (left), approximately 40 µg protein was used for blotting and approximately 400 μg protein for the co-immunoprecipitation. In the experiments with high protein expression (right), approximately 15 µg and 150 µg protein was used for blotting and co-immunoprecipitation, respectively. Blots are representative of three similar experiments. (b) Control cells (lane 1) and cells transfected with HA–R and wild-type mDRA were used to immunoprecipitate the R domain (second blot) or mDRA (third blot) and blot for the R domain (all blots). (c–e) Cells were transfected as indicated. In c, extracts were probed for protein expression. Panel d shows that CFTR (left) and the R domain (right) co-immunoprecipitate the STAS domain, but not mDRA(ΔSTAS). The blots at the left show that the STAS domain co-immunoprecipitates the R domain. In e, cells transfected with the indicated constructs were treated for 15 min with 10 µM H89 or 5 µM forskolin, as indicated, before extraction. The bands from four experiments were scanned and the measured band densities are summarized as the mean ± s.e.m. (f) PKA-phosphorylated (pRD) and non-phosphorylated (RD) recombinant GST–R domains were used to pull down the STAS domain from cell extracts.

Activation of CFTR by DRA and SLC26A6. (a) All traces and averages are colour-coded, as indicated. HEK293 cells transfected with mDRA (black), CFTR (brown), CFTR and mDRA (blue), CFTR and hDRA (purple), or CFTR and mSLC26A6 (green), were used to measure the whole-cell chloride current. Where indicated, cells were stimulated with 5 µM forskolin and inhibited with 100 μM glibenclamide. (b) A summary of four experiments under each condition in a. (c, d) Cells transfected with CFTR (c), or CFTR and mDRA (d), were used to record single-channel activity in cell-attached patches. Traces are from cells stimulated with forskolin for at least 5 min. (e, f) Averaged Po (e) and NPo (f) from four experiments. (g, h) Averaged τ0 and τc from six (CFTR) and four (CFTR and mDRA) experiments. The differences in Po, NPo and τc exceed P < 0.01, whereas the τ0 values are not different. Time and current scales are given next to the traces. (i) Expression levels of CFTR (top) and DRA (middle), and biotinylated CFTR pulled down by avidin (bottom).

STAS domain activation of CFTR requires intact STAS and R domains. All traces and averages are colour-coded, as indicated. (a) Cells were transfected with CFTR (brown), CFTR and the STAS domain of DRA (blue), CFTR and infused with MBP–STAS domain of mSLC26A6 (green), CFTR and mDRA668i (purple), or CFTR and STAS668i (light blue), and were used to measure forskolin-stimulated whole-cell current. At peak current, the cells were treated with 100 µM glibenclamide. (b) Mean ± s.e.m. of the indicated number of experiments. #P < 0.05 and *P < 0.01, compared with CFTR only. (c, d) Cells transfected with ΔR-CFTR (brown), or ΔR-CFTR and STAS domain (green), were used to measure the current before and after stimulation with forskolin (F). NS, not significant. (e–g) Single-channel activity was recorded from cells transfected with CFTR (e), CFTR and STAS (f), or CFTR and STAS668i (g). (h, i)The recorded Po (h) and NPo (i) are summarized (at least four experiments for each condition); *P < 0.05 and #P < 0.01, compared with CFTR only. (j) Expression levels of CFTR (top) and STAS (middle), and biotinylated CFTR pulled down by avidin (bottom).

Activity of STAS in vivo. (a–d) Activation of native CFTR by the STAS domain. Recombinant MBP (a, c) or MBP–STAS (30–100 µg ml−1; b, d) were infused into HEK293 cells transfected with CFTR (a, b) or duct cells freshly isolated from the parotid gland (c, d). After approximately 10 min of infusion, cells were stimulated with forskolin and then inhibited by 100 µM glibenclamide at peak current. (e) Summaries of three experiments with transfected CFTR, and five and six experiments with parotid duct cells infused with MBP and MBP-STAS, respectively. The current in a was used as the 100% control for b. The current in c was the 100% control for d. All differences from control were statistically significant (P < 0.01). (f–n) Co-localization of CFTR and SLC26A6. Sections prepared from the pancreas (f–h) and small intestine (l–n), and cells prepared from the parotid glands (i–k), were fixed and stained with rabbit polyclonal anti-SLC26A6 antibodies (f, i, l) and mouse monoclonal anti-CFTR antibodies (g, j, m). Merged images are also shown (h, k and n; SLC26A6 is shown in green, CFTR is shown in red).

A model for ductal chloride absorption and bicarbonate secretion. SLC26 transporters and CFTR assemble into a bicarbonate-transporting complex with the aid of their PDZ-binding ligands to facilitate an interaction between the CFTR R-domain and STAS domain of SLC26 transporters. This interaction switches on the activity of both proteins. The consequence of this activation is depicted in the model. Bicarbonate-secreting epithelia express different SLC26 transporters in the proximal and distal portion of the duct, with stoichiometries of two bicarbonates to one chloride and two chlorides to one bicarbonate, together with CFTR. In epithelia, an interaction between CFTR and the SLC26 transporters results in stimulation of CFTR and chloride-bicarbonate exchange. The segment of the epithelia that expresses a two-bicarbonate/one-chloride transporter absorbs the bulk of the chloride and secretes some bicarbonate, whereas the segment that expresses a two-chloride/one-bicarbonate transporter functions to concentrate the bicarbonate. Disruption of this regulation results in aberrant bicarbonate transport in CF, or to CLD and other chloride and bicarbonate transport-related diseases.